Methods of reducing the severity of mucositis

ABSTRACT

The invention provides methods of reducing the severity of mucositis, involving administration of a toll-like receptor 4 antagonist.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 10/486,455,filed Jul. 26, 2004, which is a filing under 35 U.S.C. §371 ofPCT/US02/25452, filed Aug. 12, 2002, which claims priority under 35U.S.C. §119(e) from U.S. Ser. No. 60/311,325, filed Aug. 10, 2001. Thisapplication also claims priority under 35 U.S.C. §119(e) from U.S. Ser.No. 60/680,733, filed May 13, 2005. The contents of each of the priorapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates to methods for reducing the severity ofmucositis.

Mucositis is a condition characterized by swelling, irritation, anddiscomfort of mucosal linings such as those of the gastrointestinaltract and the oral and oralpharyngeal cavities, and can result in mouthand throat sores, diarrhea, abdominal cramping and tenderness, andrectal ulcerations. This condition occurs in approximately half of allcancer patients, and is a common side effect of cancer treatmentsinvolving radiation and/or chemotherapy. The goal of these approaches tocancer treatment is to kill rapidly dividing cancer cells but,unfortunately, other rapidly dividing cells are killed by the treatmentas well, including cells that line regions such as the gastrointestinaltract, leading to mucositis. Symptoms of mucositis generally occur fiveto ten days after the start of cancer treatment, and can take two tofour weeks after cessation of treatment to clear. The incidence ofmucositis, as well as its severity, depends on factors such as the typeand duration of the cancer treatment. Mucositis occurs, for example, invirtually all patients who are treated by irradiation of the head andneck. It is also highly prevalent in patients treated with high dosechemotherapy and/or irradiation for the purpose of myeloablation, inpreparation for stem cell or bone marrow transplantation.

Mucositis adversely impacts the quality of life of cancer patients inseveral ways. For example, the mouth and throat sores of mucositis cancause significant pain and make it difficult to eat, drink, and eventake oral medication. Mucositis is also accompanied by a severe risk ofinfection, as it can lead to a breach in the otherwise protectivelinings of the oral mucosa and gastrointestinal tract, which arecolonized by a vast array of microorganisms. Further, efforts to counterthe discomforts of mucositis can lead to disruptions in cancertreatment, alterations in treatment dosages, or shifting to differentmodes of treatment. Severe mucositis can also lead to the need forparenteral nutrition or hospitalization. The development of effectiveapproaches to preventing and treating mucositis is therefore importantfor improving the care of cancer patients.

SUMMARY OF THE INVENTION

The invention provides methods of reducing the severity of mucositis(e.g., oral or gastrointestinal mucositis) in patients. The methodsinclude a step of administering to the patients a composition containingone or more compounds that block activation of toll-like receptor 4(TLR4), such as a lipid A analog, which may be within the formula:

where R¹ is selected from the group consisting of:

where each J, K, and Q, independently, is straight or branched C1 to C15alkyl; L is O, NH, or CH₂; M is O or NH; and G is NH, O, S, SO, or SO₂;

-   R² is straight or branched C5 to C15 alkyl;-   R³ is selected from the group consisting of straight or branched C5    to C18 alkyl,

where E is NH, O, S, SO, or SO₂; each A, B, and D, independently, isstraight or branched C1 to C15 alkyl;

-   R⁴ is selected from the group consisting of straight or branched C4    to C20 alkyl, and

where each U and V, independently, is straight or branched C2 to C15alkyl and W is hydrogen or straight or branched C1 to C5 alkyl;

-   R_(A) is R⁵ or R⁵—O—CH₂—, R⁵ being selected from the group    consisting of hydrogen, J′, -J′-OH, -J′-O—K′, -J′-O—K′—OH, and    -J′-O—PO(OH)₂, where each J′ and K′, independently, is straight or    branched C1 to C5 alkyl;-   R⁶ is selected from the group consisting of hydroxy, halogen, C1 to    C5 alkoxy and C1 to C5 acyloxy;-   A¹ and A², independently, are selected from the group consisting of

where Z is straight or branched C1 to C10 alkyl; or a pharmaceuticallyacceptable salt or phosphate ester thereof. One aspect of the inventionincludes phosphate esters of the above-noted formula, wherein at leastone of the hydroxyl groups of A¹ or A² can be substituted to form aphosphate ester.

An example of a Lipid A analog that can be included in the compositionsof the invention is a compound having the following structure:

or a pharmaceutically acceptable salt or phosphate ester thereof.

In a more specific example, the compound is of the following structure:

or a pharmaceutically acceptable salt or phosphate ester thereof.

Patients that can be treated according to the invention include thosewho have mucositis (e.g., oral or gastrointestinal mucositis). Inaddition, patients who do not have, but are at risk of developing,mucositis (e.g., oral or gastrointestinal mucositis) can be treatedaccording to the invention. In the latter group of patients, thetreatment can inhibit or prevent the development of mucositis.

Examples of treatments that may cause or place a patient at risk ofdeveloping mucositis (e.g., oral or gastrointestinal mucositis) areradiation therapy and chemotherapy, as described further elsewhereherein or in the background section. Patients that can be treatedaccording to the invention thus include, for example, cancer patients,as well as patients that have recently been, will shortly be, or arecurrently subject to treatment with head or neck irradiation, or stemcell or bone marrow transplantation.

According to the methods of the invention, compositions used in theinvention can be administered to a patient prior to, concurrently with,or after a treatment that has induced or places the patient at risk ofdeveloping mucositis (e.g., oral or gastrointestinal mucositis), or acombination of these approaches can be used. In an example, thecomposition is administered at the same time as, within 1-4 hours of, oron the same day as the treatment, and then for 1-3 (e.g., 1-2) daysthereafter (e.g., 1-2 times per day). Other examples of treatmentregimens are provided below.

The compositions can be administered to patients by any acceptablemanner known in the art, including topically (e.g., by gel, rinse,lozenge, cream, ointment, or patch), by intravenous infusion, orally(e.g., by tablet, capsule, lozenge, cream, ointment, or patch), rectally(e.g., by suppository, ointment, or enema), or vaginally (e.g., bycream, ointment, gel, or suppository). Also, treatment according to theinvention can be carried out in combination with other approaches totreating mucositis, including antimicrobial and palliative treatments,as is discussed further below.

Further, the invention includes compositions including the compoundsdescribed herein, formulated for administration for reducing theseverity of mucositis as described herein. As is described in detailbelow, these compositions can include the compounds in formulations suchas gels for topical administration, rinses, tablets, capsules, chewinggum, lozenges, creams, ointments, enemas, suppositories, or patches.

The invention provides several advantages. For example, in providingapproaches to reducing the severity of mucositis, an uncomfortable sideeffect of treatments such as radiation and chemotherapy, the methods ofthe invention can contribute to the well being of patients as they facethe challenges of such treatments. Further, the methods of the inventioncan decrease the incidence of infection, which is a common consequenceof mucositis. In addition, in providing increased comfort to patients,the methods of the invention can lead to increased compliance ofpatients with their therapeutic regimens, and also can contribute toincreasing the speed of their recovery.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the percent weight change of C3H/HeOuJ andC3H/HeJ mice after snout irradiation treatment. Animals were weigheddaily, the percent weight change from day 0 was calculated, and groupmeans and standard errors of the mean (SEM) calculated for each day.

FIG. 2 is a graph showing the area under the curve (AUC) calculated forthe percent weight change exhibited by snout irradiation-treatedC3H/HeOuJ and C3H/HeJ mice. This calculation was made using thetrapezoidal rule transformation. Group means were calculated and areshown with error bars representing SEM for each group. A one-way Anovatest showed a statistically significant difference between the groups(P=0.008).

FIG. 3 is a graph showing the mean serum IL-6 concentration of snoutirradiation-treated C3H/HeOuJ and C3H/HeJ mice measured by ELISAanalysis at the indicated time points.

FIG. 4 is a graph showing the mean serum TNF-α concentration of snoutirradiation-treated C3H/HeOuJ and C3H/HeJ mice measured by ELISAanalysis at the indicated time points.

FIG. 5 is a graph of epithelial histology scores for snoutirradiation-treated C3H/HeOuJ and C3H/HeJ mice. Each sample was scoredon a scale of 0-3 for epithelial cell layer damage.

FIG. 6 is a graph of connective tissue histology scores for snoutirradiation-treated C3H/HeOuJ and C3H/HeJ mice. Each sample was scoredon a scale of 0-3 for connective tissue damage.

FIG. 7 is a graph showing the mean numbers of inflammatory cells ofsnout irradiation-treated C3H/HeOuJ and C3H/HeJ mice measured at theindicated time points.

FIG. 8 is a graph showing the mean numbers of mitoses in the epithelialcell layer of snout irradiation-treated C3H/HeOuJ and C3H/HeJ micemeasured at the indicated time points.

FIG. 9 is a graph showing the mean numbers of blood vessels per 10 highpower fields of snout irradiation-treated C3H/HeOuJ and C3H/HeJ micemeasured at the indicated time points.

FIG. 10 is a graph showing the mean number of large blood vessels as apercentage of total blood vessels per 10 high power fields for C3H/HeOuJand C3H/HeJ mice measured at the indicated time points. The values areexpressed as a percentage of the total numbers of blood vessels observedin those fields.

FIG. 11 is a graph showing percent weight change of C3H/HeOuJ micetreated with the indicated amounts of eritoran, after snout irradiationtreatment. The animals were weighed daily, the percent weight changefrom day 0 was calculated, and group means and standard errors of themean (SEM) calculated for each day.

FIG. 12 is graph showing the area under the curve (AUC) calculated forthe percent weight change exhibited by snout irradiation-treatedC3H/HeOuJ mice shown in FIG. 11. This calculation was made using thetrapezoidal rule transformation. Group means were calculated and areshown with error bars representing SEM for each group. A one-way Anovatest showed no statistically significant differences between groups(P=0.261).

FIG. 13 is a graph showing the minimum number of epithelial cell layerson the dorsal surface of the tongue for snout irradiation-treatedC3H/HeOuJ mice treated with the indicated amounts of eritoran, at theindicated timepoints.

FIG. 14 is a graph showing the maximum number of epithelial cell layerson the dorsal surface of the tongue for snout irradiation-treatedC3H/HeOuJ mice treated with the indicated amounts of eritoran, at theindicated timepoints.

FIG. 15 is a graph showing the minimum number of epithelial cell layerson the ventral surface of the tongue for snout irradiation-treatedC3H/HeOuJ mice treated with the indicated amounts of eritoran, at theindicated timepoints.

FIG. 16 is a graph showing the maximum number of epithelial cell layerson the ventral surface of the tongue for snout irradiation-treatedC3H/HeOuJ mice treated with the indicated amounts of eritoran, at theindicated timepoints.

FIG. 17 is a graph showing the percent weight change of animals treatedwith eritoran (E5564) under the indicated regimens, as well asun-irradiated and placebo controls. Animals were weighed daily, thepercent weight change from day 0 was calculated, and group means andstandard errors of the mean (SEM) calculated for each day.

FIG. 18 is a graph showing the area under the curve (AUC) calculated forthe percent weight change exhibited by each animal in the study. Thiscalculation was made using the trapezoidal rule transformation. Groupmeans were calculated and are shown with error bars representing SEM foreach group. A single asterisk signifies a statistically significantdifference between a group receiving radiation and the un-irradiatedcontrols, two asterisks indicate a statistically significant differencebetween the group treated with eritoran on days 0-3, and the placebocontrols (irradiated) (P=0.030).

FIG. 19 is a graph showing the percent weight change of animals treatedaccording to the regimens indicated in the figure. Data are shown foranimals surviving until the end of the study only. Animals were weigheddaily, the percent weight change from day 0 was calculated, and groupmeans and standard errors of the mean (SEM) calculated for each day.

FIG. 20 is a graph showing the area under the curve (AUC) calculated forthe percent weight change exhibited by each animal treated, according tothe regimens noted in the graph. This calculation was made using thetrapezoidal rule transformation. Group means were calculated and areshown with error bars representing SEM for each group. A single asterisksignifies a statistically significant difference between a groupreceiving radiation and the un-irradiated controls, two asterisksindicate a statistically significant difference between the grouptreated with eritoran on days 0-3, and the placebo controls (irradiated)(P=0.041).

FIG. 21 is a graph showing mean epithelial scores and standard errors ofthe mean for each of the indicated groups.

FIG. 22 is a graph showing mean connective tissue scores and standarderrors of the mean for each of the indicated groups.

FIG. 23 is a graph showing mean inflammation scores and standard errorsof the mean for each of the indicated groups.

FIG. 24 is a graph showing mean number of mitoses per 10 hpf andstandard errors of the means for each of the indicated groups.

FIG. 25 is a graph showing the mean percent ulceration and standarderror of the mean for each of the indicated groups.

FIG. 26 is a graph showing the mean number of inflammatory cells per 10hpf and standard errors of the means for each of the indicated groups.

FIG. 27 is a graph showing the percentage of the infiltratinginflammatory cells that were neutrophils for each sample and the meanand standard deviation for each of the indicated groups.

FIG. 28 is a graph showing the percentage of the infiltratinginflammatory cells that were lymphocytes for each sample and the meanand standard deviation for each of the indicated groups.

FIG. 29 is a graph showing the percentage of the infiltratinginflammatory cells that were monocytes or macrophage for each sample andthe mean and standard deviation for each of the indicated groups.

FIG. 30 is a graph showing the number of small blood vessels per 10 hpfand the mean and standard errors of the means for each of the indicatedgroups.

FIG. 31 is a graph showing the number of medium blood vessels per 10 hpfand the means and standard errors of the means for each of the indicatedgroups.

FIG. 32 is a graph showing the number of large blood vessels per 10 hpfand the means and standard errors of the means for each of the indicatedgroups.

FIG. 33 is a graph showing the number of mast cells per 10 hpf and themeans and standard errors of the means for each of the indicated groups.

FIG. 34 is a graph showing serum TNF-α levels measured using an ELISAassay and the mean and standard error of the mean for each of theindicated groups.

FIG. 35 is a graph showing serum IL-6 levels measured using an ELISAassay and the mean and standard error of the mean for each of theindicated groups.

FIG. 36 is a graph showing serum SAA levels measured using an ELISAassay and the mean and standard error of the mean for each of theindicated groups.

DETAILED DESCRIPTION

The present invention provides methods of reducing the severity ofmucositis (e.g., oral or gastrointestinal mucositis). The methods can beused to treat patients who already have mucositis. In addition, themethods can also be carried out with patients who do not have, but areat risk of developing mucositis (e.g., cancer or other patientsscheduled to receive, currently receiving, or previously treated withradiation and/or chemotherapy). In the latter group of patients, whichdo not yet have mucositis, treatment according to the invention canreduce the severity of mucositis resulting from their cancer treatment,inhibit the development of mucositis, or prevent mucositis.

The invention is based on the discovery that blocking activation oftoll-like receptor 4 (TLR4) provides beneficial therapeutic effects inthe reduction of severity of mucositis, as described herein. TLR4 is areceptor for endotoxin, or lipopolysaccharide (LPS), which is shed fromthe cell walls of growing and dying bacteria and has been associatedwith the induction of inflammatory responses. According to the presentinvention, TLR4 receptor activation is blocked by administration of aTLR4 antagonist, leading to beneficial effects in the reduction ofseverity of mucositis. In addition to blocking endotoxin, treatmentaccording to the invention may block the effects of heat shock proteins(HSP's) in mucositis. In particular, such proteins, which are stressinducible proteins, may be induced during stress including radiationtherapy and chemotherapy. HSP60, 70, or 90 may be endogenous ligands ofTLR4, and thus may play a role in the mucositis induced by radiationtherapy.

TLR4 antagonists used in the methods of the invention can be, forexample, analogs of the lipid A region of LPS, such as lipid A analogsthat are within the formula set forth above, in the Summary of theInvention. An example of a Lipid A analog that can be included in thecompositions of the invention is a compound having the followingstructure:

or a pharmaceutically acceptable salt or phosphate ester thereof.

In a more specific example, the compound is of the following structure:

or a pharmaceutically acceptable salt or phosphate ester thereof. Thiscompound, is known as eritoran (also known as compound E5564, compound1287, and SGEA) and is described in U.S. Pat. No. 5,935,938.

Other examples of compounds that can be used in the invention includethe following:

and a pharmaceutically acceptable salt or phosphate ester thereof.

Additional TLR4 antagonists that can be used in the invention include,for example, compound B531 (U.S. Pat. No. 5,530,113), as well as othercompounds described in the following patents: U.S. Pat. No. 5,935,938;U.S. Pat. No. 5,612,476; U.S. Pat. No. 5,756,718; U.S. Pat. No.5,843,918; U.S. Pat. No. 5,750,664; U.S. Pat. No. 6,235,724; U.S. Pat.No. 6,184,366; and U.S. Pat. No. 5,681,824. Methods for making thesecompounds are also described in these documents. Additional methods formaking such drugs are described, for example, in WO 02/94019.

According to the methods of the invention, a TLR4 antagonist isadministered to a patient before, during, and/or after treatment with atherapy that causes mucositis (e.g., oral or gastrointestinal mucositis)or puts the patient at risk of developing such mucositis. As is notedabove, such treatments include radiation and chemotherapy, which act byblocking the growth of rapidly dividing cells, such as cancer cells andepithelial cells that line the surfaces of the gastrointestinal,respiratory, and genitourinary tracts. Specific examples of treatmentsthat can lead to mucositis include radiation treatment (e.g., headand/or neck, whole body, targeted, and/or hyperfractionated radiation),as well as chemotherapeutic regimens used in the treatment of, or asadjuvant treatments for, conditions such as breast cancer, colon cancer,gastric cancer, genitourinary (e.g., bladder, prostate, or testicular)cancer, gynecologic (e.g., cervical, endometrial, ovarian, or uterine)cancer, head and neck/esophageal cancer, leukemia, lung (small cell ornon small-cell) cancer, lymphoma (Hodgkin's or non-Hodgkin's), melanoma,multiple myeloma, pancreatic cancer, and sarcoma.

As is known in the art, cancers such as these can be treated usingapproaches involving immunotherapy by use of agents such as, forexample, rituximab, cetuximab, or bevacizumab, alone or in combinationwith chemotherapy or radiation therapy. In other examples,chemotherapeutic approaches that may induce mucositis include thoseutilizing (either as single agents or in combinations) platinumderivatives such as carboplatin, cisplatin, and oxaplatin; mitosisinhibitors such as paclitaxel, docetaxel, vinorelbine, vincristine, andvinblastine; topoisomerase inhibitors such as etoposide, irinotecan, andtopotecan; antimetabolites such as gemcitabine, capecitabine,fludarabine, methotrexate, 5-fluorouracil, cladribine, pentostatin, andcytarabine; DNA synthesis inhibitors such as doxorubicin, epirubicin,idarubicin, daunorubicin, bleomycin, mechlorethamine, and mitoxantrone;alkylating agents such as cyclophosphamide, ifosfamide, and melphalancarmustine; hormonal oncologics such as estramustine; and agents havingother or unknown mechanisms such as dacarbazine. Use of these and otherapproaches to treating cancer is well known to those of skill in theart.

TLR4 antagonists such as those noted above can be administered usingstandard methods including, for example, topical approaches andintravenous infusion. The particular approach and dosage used for aparticular patient depends on several factors including, for example,the type of cancer treatment, the location(s) of any discomfort, and thegeneral health of patient. Based on factors such as these, a medicalpractitioner can select an appropriate approach.

Treatment according to the invention can begin prior to cancer treatment(e.g., 1-2 days or up to 1 week prior to cancer treatment), at or nearthe same time as cancer treatment (e.g., simultaneously with, within 1-4hours of, or on the same day as cancer treatment), or shortly after thecessation of cancer treatment (e.g., within 1-4 days of cessation,and/or prior to or upon appearance of symptoms). Treatment can then bemaintained, for example, until any symptoms of mucositis havesubstantially cleared or the risk of developing such symptoms haspassed. Thus, treatment started before or at or near the same time ascancer treatment can be maintained, e.g., for 1-3, e.g., 1-2 days. Inother examples, treatment is maintained for 1-4 or 2-3 weeks followingthe cessation of cancer treatment, as determined to be appropriate byone of skill in the art. In specific examples, the treatment accordingto the present invention is carried out prior to cancer treatment only;prior to and concurrently with cancer treatment only; prior to,concurrently with, and after cessation of cancer treatment; concurrentlywith cancer treatment only; concurrently with and after cessation ofcancer treatment only; after cessation of cancer treatment only; orprior to and after cessation of cancer treatment only. Further,treatment according to the methods of the invention can be altered,stopped, or re-initiated in a patient, depending on the status of anysymptoms of mucositis. Treatment can be carried out at intervalsdetermined to be appropriate by those of skill in the art. For example,the administration can be carried out 1, 2, 3, or 4 times/day.

In the case of patients having or at risk of developing mucositis in theoral cavity, a TLR4 antagonist, as described herein, can be administeredto the oral cavity in the form of a gel, paste, spray, cream, ointment,or patch that is applied to affected or at risk areas. Such patients canalso be treated by the use of an oral rinse, chewing gum, or lozengeincluding the drug. The drug can be administered to patients affected inrectal or vaginal areas by use of formulations in the form of gels,creams, ointments, suspensions, or suppositories. Further,administration can be by use of an enema. In another example, in thecase of patients affected in the nasal cavity, the drug can beadministered by topical administration, as described herein, or byinhalation of the drug (see, e.g., U.S. Pat. No. 6,683,063). In otherapproaches, the drug can be administered by injection (e.g., localinjection), or by infusion (intravenous or intra-arterial), as discussedfurther below.

Formulation of drug compounds for use in the modes of administrationnoted above (and others) are known in the art and are described, forexample, in Remington's Pharmaceutical Sciences (18^(th) edition), ed.A. Gennaro, 1990, Mack Publishing Company, Easton, Pa. (also see, e.g.,M. J. Rathbone, ed., Oral Mucosal Drug Delivery, Drugs and thePharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y., U.S.A., 1996;M. J. Rathbone et al., eds., Modified-Release Drug Delivery Technology,Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y.,U.S.A., 2003; Ghosh et al., eds., Drug Delivery to the Oral Cavity,Drugs and the Pharmaceutical Sciences Series, Marcel Dekker, Inc., N.Y.,U.S.A., 2005; and Mathiowitz et al., eds., Bioadhesive Drug DeliverySystems, Drugs and the Pharmaceutical Sciences Series, Marcel Dekker,Inc., N.Y., U.S.A., 1999.

All patients, and in particular those affected (or at risk) in internalregions that are not readily accessible for topical administration, canbe treated by a systemic approach, such as intravenous infusion. Thisapproach to administration may be particularly convenient in the case ofpatients who already have a catheter in place for the administration ofchemotherapeutic or other drugs. Examples of such approaches, in whichthe drug administered is eritoran (see above) and the indicated amountsof the drug are based on an assumed average weight of a subject of 70kg, are as follows. In a first example, the drug can be administered ata low dosage by continuous intravenous infusion. As a specific example,the drug can be administered continuously at a rate of 10-500 (e.g.,50-400 or 100-200) μg/hour over the course of the treatment. In anotherexample, in which a patient requires longer-term care, the drug can beadministered intermittently (e.g., every 12-24 hours) at a dosage of,for example, 0.1-20 (e.g., 1-8, 2-7, 3-6, or 4-5) mg/hour for 2-6 (e.g.,approximately 4) hours. In a variation of this approach, the initial orloading dose is followed by maintenance doses that are less than (e.g.,half) the loading dose or by continuous infusion as described above inthe first example. The duration of such treatment can be determined bythose of skill in the art, based on factors such as, for example, theseverity of the condition and the observation of improvements.Additional details concerning the use of infusion to administer TLR4antagonists, such as eritoran, are provided in US-2003-0105033-A1 (bolusor intermittent infusion) and WO 00/41703 (continuous infusion), thecontents of each of which are incorporated herein by reference.

When administering the compound eritoran by intravenous infusion, it ispreferable to use devices and equipment (e.g., catheters, such ascentral or peripheral venous catheters, tubing, drip chambers, flashbackbulbs, injection Y sites, stopcocks, and infusion bags) that arecompatible with the drug. In particular, catheters including achlorhexidine-based antimicrobial coating have been found to disrupt thesize of the micelles of the drug that are formed during formulation,leading to inadequate concentrations in blood. Thus, it is preferable touse devices and equipment that have, for example, anon-chlorhexidine-based antimicrobial coating, such as an antimicrobialcoating that includes one or more other antibiotics, such as rifampin orminicyclin.

The invention also includes kits that include one or more TLR4antagonists (e.g., a Lipid A analog as described above, e.g., thecompound eritoran) and instructions to use the drug in the methodsdescribed herein. The kits can also optionally include devices orequipment used in administration (e.g., a catheter lacking achlorhexidine coating) and/or a solution for administering the drug,such as a 5% dextrose (e.g., glucose) solution.

The methods of the invention can be used alone or in conjunction withother approaches to reducing the severity of mucositis. For example, themethods of the invention can be carried out in combination withantimicrobial or antifungal therapies, e.g., therapies involvingadministration of antibiotics such as nystatin, amphotericin, acyclovir,valacyclovir, clotimazole, and fluconazole. As a specific example ofsuch treatment, patients with head and neck cancer receivingradiotherapy have colonization of the oropharyngeal region withgram-negative bacteria. Selective decontamination of the oral cavitywith anti-microbial agents has the benefit of reducing oral mucositisassociated with radiation therapy, but there may be limitations to thebeneficial effects of such treatment. Anti-microbial therapy can killbacteria, but cannot reduce endotoxin, and indeed may actually increaseendotoxin. As endotoxin is a potent mediator of inflammation, it maycontribute to the aggravation of mucositis and, thus, co-treatment withan antiendotoxin compound (e.g., a Lipid A analog, such as eritoran) andantibiotics can be used as a more effective approach to treating oralmucositis in such patients, according to the invention.

The methods of the invention can also be used in conjunction withpalliative therapies including the use of topical rinses, gels, orointments that include lidocaine, articaine, and/or morphine, as well asother analgesic or anti-inflammatory agents. Specific examples of otheragents and approaches that can be used in combination with TLR4antagonists, according to the methods of the invention, include thefollowing: palifermin (recombinant keratinocyte growth factor; rHuKGF;Kepivance™; Amgen) and AES-14 (uptake-enhanced L-glutamine suspension)(Peterson, J. Support Oncol. 4(2 Suppl. 1)9-13, 2006); oral cryotherapy,low-level laser therapy, chlorhexidine, amifostine, hematologic growthfactors, pentoxifylline, and glutamine (Saadeh, Pharmacotherapy25(4):540-554, 2005); amifostine, antibiotic paste or pastille,hydrolytic enzymes, ice chips, benzydamine, calcium phosphate, honey,oral care protocols, povidone, and zinc sulphate (Worthington et al.,Cochrane Database Syst. Rev. 2:CD000978, 2006); flurbiprofen (e.g.,administered as a tooth patch; Stokman et al., Support Care Cancer13(1):42-48, 2005); diphenhydramine, magnesium hydroxide/aluminumhydroxide, nystatin, and corticosteroids (Chan et al., J. Oncol. Pharm.Pract. 11(4):139-143, 2005); oral transmucosal fentanyl citrate (e.g.,administered in the form of a lozenge; Shaiova et al., Support CareCancer 12(4):268-273, 2004); clonazepam (e.g., in the form of a tablet;Gremeau-Richard et al., Pain 108(102):51-57, 2004); capsaicin (e.g., inthe form of a lozenge; Okuno et al., J. Cancer Integr. Med.2(3):179-183, 2004); ketamine (e.g., in the form of an oral rinse;Slatkin et al., Pain Med. 4(3):298-303, 2003); andgranulocyte-macrophage colony-stimulating factor (GM-CSF)/granulocytecolony-stimulating factor (G-CSF), laser light therapy, and glutaminesupplements (Duncan et al., Aliment. Pharmacol. Ther. 18(9):853-874,2003).

The present invention is based, in part, on the following experimentalresults.

Example I 1. Introduction 1.1 Rationale

Two strains of C3H mice (C3H/HeJ and C3H/HeOuJ) differ from one anotherby the presence or absence of the LPS receptor TLR4 (present in theC3H/HeOuJ strain). C3H/HeJ mice are more sensitive to the lethal effectsof total body radiation, but do not develop oral mucositis to the sameextent as do the C3H/HeOuJ mice after a localized acute radiation to thesnout. The mechanistic basis for these differences is not understood.

1.2 Acute Snout Radiation Model

The acute mouse snout radiation model in mice has been used to determinethe radioprotective properties of experimental compounds. The course oforal mucositis in this model is well defined and results in peakmucositis 10-12 days following radiation. The acute model has littlesystemic toxicity, resulting in few radiation-induced animal deaths. Inthe present study, we used a dose of 30 Gy to induce oral mucositis.

2. Study Objective and Summary 2.1 Study Objective

The objective of the study described below was to evaluate the effect oflocalized acute radiation on the severity and duration of oral mucositison two strains of mice. Wild type C3H/HeOuJ mice were compared to theendotoxin resistant strain C3H/HeJ. Mucositis was induced using an acuteradiation dose of 30 Gy directed to the Mouse snout. At several timepoints after radiation, groups of four mice of each strain weresacrificed. At the time of sacrifice, the tongues were removed anddissected into three pieces. The anterior third of each tongue was fixedin formalin for subsequent histological analysis. The middle third ofeach tongue was extracted to provide mRNA for analysis of cytokineexpression levels. The posterior portion of each tongue was flash frozenin liquid nitrogen for future analysis. At the time of sacrifice, bloodwas taken from each animal and serum was prepared for subsequentcytokine analysis. This study focused on the pro-inflammatory cytokinesTNF-α and IL-6.

2.2 Study Summary

A total of sixty-four (64) mice were used. Fifty-six (56) mice (28 eachC3H/HeOuJ and 28 C3H/HeJ) were given a single dose of 30 Gy radiationdirected to the snout on day 0. In addition, eight (8) mice (4 C3H/HeOuJand 4 C3H/HeJ) were used as the no radiation control animals. Animalswere sacrificed and blood and tissue taken according to the scheduledescribed in Table 1.

TABLE 1 Histological and cytokine comparison of the effects of ionizingradiation of the oral mucosa of C3H/HeJ and C3H/HeOuJ mice Hour/Day 0 2H 6 H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Randomize animals X Weigh, Recordsurvival X X X X X X X X X X X X X X X Irradiate all animals 30 Gy tosnout X Sacrifice 4 animals/group (1 and 2) X X X X X X X dissect oralmucosa Sacrifice 4 animals/group (3 and 4) X dissect oral mucosa Obtainserum from each animal X X X X X X X

3. Study Design

Sixty-four (64) mice (32 C3H/HeOuJ and 32 C3H/HeJ) were used. The micewere randomized into four (4) groups of either 28 animals (groups 1 and2), the radiated groups, or 4 animals (groups 3 and 4), theun-irradiated control groups, as described in Table 2.

TABLE 2 Animal allocation by experimental group. Mouse Radiation GroupStrain N 30 Gy 1 C3H/HeOuJ 28 YES WT 2 C3H/HeJ 28 YES Mutant 3 C3H/HeOuJ4 NO WT 4 C3H/HeJ 4 NO Mutant

Every day for the period of the study (day 0 to day 14), each animal wasweighed. Animals in groups 1 and 2 received a single dose of 30 Gyradiation focused on the snout on day 0. A lead shield protected theremainder of the animal body. At 2 hours, 6 hours, 24 hours (1 day), 3days, 6 days, 10 days, and 14 days after radiation, 4 animals fromgroups 1 and 2 were sacrificed and blood and tissue were collected asdescribed below. Animals in groups 3 and 4 were sacrificed, the tonguesdissected, and blood collected on day 1. The tongues from each animalwere dissected into 3 pieces (anterior, middle, and posterior) and eachtongue was fixed in formalin. Mucositis was assayed by histologicalanalysis of hematoxylin and eosin (H&E) stained sections of the formalinfixed tongues. Mucositis scoring was done in a blinded manner accordingto a validated scale. Serum samples were assayed for the cytokines TNF-αand IL-6 using a standard ELISA assay.

4. Material and Methods 4.1 Animals

C3H/HeOuJ and C3H/HeJ mice (Jackson Laboratories), aged 5 to 6 weekswith body weights of 22.3 g, were used. Animals were individuallynumbered using an ear punch and housed in small groups of approximately5 animals per cage. Animals were acclimatized prior to studycommencement. During this period of at least 2 days, the animals wereobserved daily in order to reject animals that presented in poorcondition.

4.2 Housing

The study was performed in animal rooms provided with filtered air at atemperature of 70° F.+/−5° F. and 50%+/−20% relative humidity. Animalrooms were set to maintain a minimum of 12 to 15 air changes per hour.The room was on an automatic timer for a light/dark cycle of 12 hours onand 12 hours off with no twilight. Bed-O-Cobs® bedding was used, and waschanged a minimum of once per week. Cages, tops, bottles, etc. werewashed with a commercial detergent and allowed to air dry. Prior to use,these items were wrapped and autoclaved. A commercial disinfectant wasused to disinfect surfaces and materials introduced into the hood.Floors were swept daily and mopped a minimum of twice weekly with acommercial detergent. Walls and cage racks were sponged a minimum ofonce per month with a dilute bleach solution. A cage card or label withthe appropriate information necessary to identify the study, dose,animal number, and treatment group was placed on all cages. Thetemperature and relative humidity was recorded during the study, and therecords retained.

4.3 Diet

Animals were fed with a Labdiet® 5001 chow and water was provided adlibitum.

4.4 Animal Randomization and Allocations

Mice were randomly and prospectively divided into four (4) treatmentgroups prior to irradiation. Each animal was identified by an ear punchcorresponding to an individual number. A cage card was used to identifyeach cage or label marked with the study number, treatment group number,and animal numbers.

4.5 Radiation

Machine calibration was checked within two weeks of the onset of thestudy. A single dose of radiation (30 Gy/dose) was administered to allanimals in groups 1 and 2 on day 0. Radiation was generated with a 160kilovolt potential (15-ma) source at a focal distance of 50 cm, hardenedwith a 0.35 mm Cu filtration system. Irradiation was done at a rate of121.5 cGy/minute. Animals were anesthetized prior to radiation, andplaced under lead shielding such that only the snout was exposed

4.6 Tissue and Blood Collection and Analysis 4.6.1 Animal Sacrifice andTissue Collection

Animals in groups 3 and 4 were the un-irradiated control animals. Themeasurements from these animals provided a baseline control for all theirradiated samples in this study. The 4 animals in each of groups 3 and4 were sacrificed on day 1.

Animals from groups 1 and 2 were sacrificed at several time pointsduring the course of the study. At each time point, 4 animals per groupwere sacrificed. The time points were 2 hours, 6 hours, 24 hours, 3days, 6 days, 10 days, and 14 days after radiation.

At the time of sacrifice, the tongues were removed and dissected intothree pieces. The anterior third of each tongue was fixed in formalinfor subsequent histological analysis. The middle third of each tonguewas extracted to provide mRNA for analysis of cytokine expressionlevels. The posterior section of each tongue was flash frozen in liquidnitrogen and stored for future analysis.

At the time of sacrifice, approximately 1 mL of blood was taken fromeach animal and serum was prepared for subsequent cytokine analysis.This study focused on the pro-inflammatory cytokines TNF-α and IL-6.

4.6.2 Cytokine ELISA

Enzyme linked immunosorbent assays (ELISAs) were performed for cytokinesTNF-α and IL-6 using kits purchased from R and D systems. These kitswere used in accordance with the manufacturer's instructions. Alldeterminations were made in duplicate on serum samples stored at −80° C.If insufficient serum had been collected to run both IL-6 and TNF-α,samples were diluted 1:2 or 1:4, and run in duplicate in both assays.All assays were performed using 50 μL of sample per well.

4.6.3 Histology

Histological samples were fixed in 10% formaldehyde in saline andprocess for paraffin histology using standard techniques. Slides werestained with hematoxylin and eosin (H&E) and reviewed by a boardcertified pathologist.

4.7 Assessment of Results

Statistical differences between treatment groups were determined usingOne Way ANOVA. Body weights were evaluated for differences between thetreatment groups.

5. Results and Discussion 5.1 Survival

A total of 6 deaths occurred on day 10. These were equally distributedbetween the C3H/HeOuJ and C3H/HeJ groups (3 deaths in each group) and,as a result of this, only one animal in each group was sacrificed on day14. Additional animals were subsequently irradiated in order to provideadditional animals to provide data for the day 14 time-point.

5.2 Weights (FIGS. 1 and 2)

The mean percentage weight change for each group is shown in FIG. 1. Theweight change data show that both groups of animals lost approximately5% of their starting body weight by day post irradiation, then gainedweight until day 6. From day 6 until day 13, the C3H/HeJ mice maintainedtheir weight between no gain and 5% increase relative to their startingweight. The C3H/HeOuJ lost approximately 10% of their body weightbetween days 7 and 9, and did not gain weight before day 13. To evaluatethe differences between the two groups, the area under the curve (AUC)for each individual animal was calculated and the differences wereevaluated using a One-Way ANOVA analysis. The mean AUC data is shown inFIG. 2. The One-Way ANOVA analysis showed that there was a statisticallysignificant difference between the groups (P=0.008).

5.3 Serum Cytokine Levels

Serum levels of cytokines IL-6 and TNF-α were evaluated by ELISA.

5.3.1 Serum IL-6 Concentrations

In un-irradiated C3H/HeOuJ mice, the mean serum concentration of IL-6was 1.0 pg/mL. This level increased to 88.8 pg/mL at 6 hours postradiation, before falling to 12.0 pg/mL on day 3 following radiation andincreasing to a peak level of 122.7 pg/mL on day 6. Days 10 and 14showed a gradual decline from the peak levels seen on day 6. Inun-irradiated C3H/HeJ mice, the mean serum concentration of IL-6 was13.8 pg/mL. All other readings were between 25 and 42 pg/mL with theexception of the day 10 time-point, when serum IL-6 concentrationsincreased to 69.4 pg/mL. These data are shown in FIG. 3.

5.3.2 Serum TNF-α Concentrations

The mean serum TNF-α concentration in un-irradiated C3H/HeOuJ mice was48.0 pg/mL. There were 2 peaks in serum TNF-α levels in these mice, oneafter 2 hours (168.7 pg/mL) and one at day 10 (410.2 pg/mL). Attime-points between these 2 peaks, serum TNF-α concentrations were closeto the levels seen in un-irradiated C3H/HeOuJ mice (31.6 pg/mL to 87.2pg/mL). On days 10 and 14, the levels were lower than in theun-irradiated controls (5.7 pg/mL and 6.6 pg/mL respectively). InC3H/HeJ mice, un-irradiated control mice had mean serum TNF-αconcentrations of 109.3 pg/mL. Subsequent post-irradiation readings weregenerally lower than this, ranging from 14.2 pg/mL at 6 hours postradiation to 133.8 pg/mL on day 10. These data are shown in FIG. 4.

5.4 Tongue Histology

Parts of each tongue were processed for routine hematoxylin and eosin(H&E) histology. These slides were then reviewed by a board certifiedpathologist and scored for epithelial and connective tissue pathology ona scale of 0-3, epithelial mitoses, percent ulceration, skeletal muscledamage, number of inflammatory cells per 10 high powered fields(including differential cell type analysis), and the number of small,medium, and large blood vessels.

5.4.1 Histological Score

The epithelium and connective tissue regions of each sample were eachgiven separate scores. The scores for the epithelium are shown in FIG.5. The mean epithelial histological score for C3H/HeOuJ mice that hadnot been irradiated was 0, and this was also the case for allpost-radiation time points except day 1, when the mean score was 0.25,and days 6 and 10, when the mean score was 2. In C3H/HeJ mice, the meanepithelial histological score was 0 at all time points except day 6,when the score was 0.75. The data for the mean connective tissuehistological scores is shown in FIG. 6. The mean connective tissuehistological score for C3H/HeOuJ mice that had not been irradiated was2. This score dropped to 0 at 2 hours post-radiation, increased to ascore of 1 on day 1 post radiation, before dropping to 0 on day 3 andincreasing to 1.5 on days 10 and 14. In C3H/HeJ mice, the meanepithelial histological score was 1.25 in mice that had not beenirradiated, dropping to 0.25 at 2 hours post radiation before increasinggradually to 1.25 on day 10. In the connective tissue, the histologicalscore was as high or higher in the control un-irradiated mice than atany time following radiation, in both C3H/HeOuJ and C3H/HeJ mice. Thereasons for this are currently unknown.

5.4.2 Inflammation

The mean number of inflammatory cells per ten high powered fields foreach strain of mouse at each time point was calculated and the resultsare shown in FIG. 7. The numbers of cells seen in the connective tissueof un-irradiated animals was higher than expected in both strains ofmice, and was lower at all post irradiation time-points in the C3H/HeJmice. In the C3H/HeOuJ mice, the numbers of inflammatory cells seen atmost time points was also lower than those observed in un-irradiatedcontrols, except for day 10, when the number of cells was approximately2 times higher than the un-irradiated controls (and about 10 timeshigher than the 2 hour and 6 hour time-points) and on day 14, when thenumbers observed were about 50% higher than the un-irradiated controls.As for the connective tissue histology scores, the reasons for theunexpectedly high numbers seen in the un-irradiated animals are unknown.In all cases, the bulk of the infiltrate was composed of lymphocytes,with monocytes and macrophage accounting for almost all of thenon-lymphocytic cells most animals. Significant numbers ofpolymorphonucleocytes (PMNs or neutrophils) were only seen in threeanimals from the day 10 time-point (1 OuJ and 2 HeJ).

5.4.3 Epithelial Cell Mitoses

The number of mitotic figures seen in the epithelial cell layer wascounted and the mean number of mitoses per ten high power fields foreach strain at each time point is shown in FIG. 8. The number of mitoticfigures counted in the epithelial cell layer of C3H/HeOuJ mouse tongueswas generally low, with a mean of 0.4 in un-irradiated mice and numberslower than this at all time points except day 6, when a mean of 2.75 wasobserved. In C3H/HeJ mice the numbers of mitoses seen in un-irradiatedmice was lower than in C3H/HeOuJ mice with a mean of 0.1. However, thisincreased to 0.4 by day 10 post radiation.

5.4.4 Blood Vessels

The number of blood vessels per ten high power fields was counted foreach sample and the mean number for each strain of mouse at each timepoint calculated. These data are shown in FIG. 9. The number of bloodvessels per 10 high power fields was 26.6 in un-irradiated C3H/HeOuJmice and 27.2 in un-irradiated C3H/HeJ mice. In C3H/HeOuJ mice, thenumber of blood vessels had apparently dropped to 4.5 by 2 hours postradiation, rising to 20.6 on day 1, before falling to 8.5 on day 3, andincreasing on days 6 and 10, before reaching a peak of 33.7 on day 14.This represents an increase of 27% relative to the un-irradiatedcontrols and 648% relative to the 2 hour time point. It is interestingto note that the un-irradiated control animals, sacrificed on day 1,have similar levels to the day 1 time-point. In the C3H/HeJ mice, thenumber of blood vessels was generally close to the un-irradiatedcontrols, reaching a minimum of 18.3 at 2 hours post radiation, and amaximum of 33.1 on day 6 post radiation. To evaluate the qualitativechanges in the blood vessels, the numbers of large blood vessels per 10high power fields were evaluated and the resulting numbers expressed asa percentage of the total number of vessels seen in the same 10 highpower fields. The results of this analysis are shown in FIG. 10 andindicate that the number of large blood vessels seen in the C3H/HeOuJmice increased from a mean of 9.2% in the un-irradiated control mice, toa peak of 26.5% on day 1 (24 hours post radiation), and declined duringthe remainder of the study. The C3H/HeJ mice had a slightly highercontrol level of 13.2% in the un-irradiated mice, which increased to apeak of 18.7% on day 6 post radiation, and fell to levels below controlson days 10 and 14 post radiation.

6. Conclusions

1. C3H/HeOuJ mice showed greater weight loss than the C3H/HeJ miceduring this study, and the differences observed were statisticallysignificant when evaluated with a one-way ANOVA test (P=0.008).

2. Analysis of serum cytokine levels showed that the un-irradiatedC3H/HeJ control mice had higher levels than their C3H/HeOuJcounterparts, but that the C3H/HeOuJ mice showed greater increases inserum cytokines following radiation than C3H/HeJ, with peak levels ofboth IL-6 and TNF-α being seen on day 6 post irradiation.

3. Histologically, very little change was seen in C3H/HeJ mice.C3H/HeOuJ mice showed a significant disturbance of the epithelium ondays 6 and 10 following radiation. Histological scores for theconnective tissue were high in the un-irradiated control C3H/HeOuJ miceand declined at 2 hours to 6 days post radiation, returning to nearcontrol levels at days 10 and 14 post radiation.

4. The numbers of inflammatory cells present showed little change in theC3H/HeJ mice but increased to a peak on day 10 post radiation inC3H/HeOuJ mice, coinciding with the tissue peak tissue cytokine levelsin these animals. Infiltrates were predominantly lymphocytic in nature.

5. The number of mitoses observed in the epithelial cell layer showed aslight increase in C3H/HeJ mice, peaking on day 10, while a significantspike in mitotic activity was noted on day 6 in the C3H/HeOuJ mice.

6. In the analysis of the number and size of blood vessels observed, fewchanges were noted in the C3H/HeJ mice, while the C3H/HeOuJ mice showeda decrease in the number of blood vessels immediately followingradiation (2 and 6 hours post radiation), combined with an overallincrease at the later time points (days 10 and 14 post radiation). Anincrease in the percentage of large blood vessels was noted 24 hourspost radiation in C3H/HeOuJ mice.

Example II 1. Introduction 1.1 Rationale

As noted above in Example I, two strains of C3H mice (C3H/HeJ andC3H/HeOuJ) differ from one another by the presence or absence of the LPSreceptor TLR4 (present in the C3H/HeOuJ strain). In the experimentsdescribed above, it is established that the C3H/HeOuJ strain issusceptible to oral mucositis induced by focal radiation to the snout,while the C3H/HeJ strain is relatively resistant to radiation inducedmucositis. Evaluation of the pro-inflammatory cytokines in these animalsshowed that the induction of these cytokines via the LPS receptor TLR4in the C3H/HeOuJ mice may play a role in the development of oralmucositis. The purpose of the study described below was to evaluate acompound that blocks stimulation of TLR4 (eritoran) in the murine modelof oral mucositis.

1.2 Acute Snout Radiation Model

The acute mouse snout radiation model in mice has been used to determinethe radio-protective properties of experimental compounds. The course oforal mucositis in this model is well defined and results in peakmucositis 10-12 days following radiation. The acute model has littlesystemic toxicity, resulting in relatively few radiation induced animaldeaths. In this study, a dose of 30 Gy was used to induce oralmucositis.

2. Study Objective and Summary 2.1 Study Objective

The objective of the study described below was to examine the effects oferitoran administered subcutaneously on the severity and duration oforal mucositis induced by radiation. Mucositis is induced using an acuteradiation dose of 30 Gy directed to the mouse snout. At several timepoints after radiation, groups of four mice from each treatment groupwere sacrificed. At the time of sacrifice, the tongues were removed anddissected into three pieces. The anterior third of each tongue was fixedin formalin for subsequent histological analysis. The middle third ofeach tongue was extracted to provide mRNA for analysis of cytokineexpression, and the posterior portion of each tongue was flash frozen inliquid nitrogen and stored for future analysis. At the time ofsacrifice, blood was taken from each animal and serum was prepared forsubsequent cytokine analysis.

2.2 Study Summary

A total of fifty-four (54) animals were used in this study. Forty-eight(48) C3H/HeOuJ mice were divided into 3 groups of 16 animals per group(groups 1-3). An additional 6 animals were put into a separate controlgroup (group 4) as described in Table 2.

3. Study Design

Fifty-four (54) male C3H/HeOuJ mice aged 6-7 weeks and weighingapproximately 22 g were used. There were three (3) treatment groups ofsixteen (16) animals each, and a control group of six (6) animals thatreceived no radiation. All animals had a jugular cannula inserted intothe left jugular vein on day −3. Beginning on day 0, animals in groups 1and 4 were dosed twice a day by injection via cannula with placebo.Animals in group 2 were dosed with 2 IV injections of eritoran at, 1mg/kg daily, starting 2 hours or less before radiation on day 0 andcontinuing until day 10. Animals in group 3 were dosed with 2 IVinjections of eritoran at 10 mg/kg daily, starting 2 hours or lessbefore radiation on day 0 and continuing until day 10. Animals in groups1, 2, and 3 were given a single dose of 30 Gy radiation directed to thesnout on day 0. The 6 animals in group 4 were used as the no radiationcontrol animals (see Table 2). Eight animals in each of groups 1, 2, and3 were sacrificed and blood and tissue taken according to the scheduledescribed in Table 2.

TABLE 2 Allocation by experimental group. Number of Group animals StrainTreatment Radiation Sac points 1 16 male C3H/HeOuJ placebo IV bid 30 Gyto snout 8 on day 6, 8 on day 10 2 16 male C3H/HeOuJ eritoran 1 mg/kg IVbid 30 Gy to snout 8 on day 6, 8 on day 10 3 16 male C3H/HeOuJ eritoran10 mg/kg IV 30 Gy to snout 8 on day 6, bid 8 on day 10 4  6 maleC3H/HeOuJ placebo IV bid none 6 on day 10

4. Material and Methods 4.1 Animals

C3H/HeOuJ mice (Jackson Laboratories), aged 5 to 6 weeks with bodyweight of 21.3 g, were used. Animals were individually numbered using anear punch and individually housed. Animals were acclimatized prior tostudy commencement. During this period of at least 2 days, the animalswere observed daily in order to reject animals that presented in poorcondition.

4.2 Housing

The study was performed in animal rooms as described above in section4.2 of Example I.

4.3 Diet

Animals were fed with Labdiet® 5061 sterile irradiated rodent chow andwater was provided ad libitum.

4.4 Animal Randomization and Allocations

Mice were randomly and prospectively divided into three (3) treatmentgroups prior to irradiation. Each animal was identified by an ear punchcorresponding to an individual number. A cage card was used to identifyeach cage or label marked with the study number, treatment group number,and animal numbers.

4.5 Radiation

Machine calibration was checked within two weeks of the onset of thisstudy. A single dose of radiation (30 Gy/dose) was administered to allanimals in groups 1 and 2 on day 0. Radiation was generated with a 160kilovolt potential (15-ma) source at a focal distance of 50 cm, hardenedwith a 0.35 mm Cu filtration system. Irradiation was done at a rate of121.5 cGy/minute. Animals were anesthetized prior to radiation, andplaced under lead shielding such that only the snout is exposed

4.6 Tissue Collection and Analysis 4.6.1 Histology

Histological samples were fixed in 10% formaldehyde in saline andprocess for paraffin histology using standard techniques. Slides werestained with hematoxylin and eosin (H&E).

4.7 Assessment of Results

Statistical differences between treatment groups were determined usingOne Way ANOVA. Body weights were evaluated for differences between thetreatment groups.

5. Results and Discussion 5.1 Weights (FIGS. 11 and 12)

The mean percentage weight gain for each group for each day of the studyis shown in FIG. 11. The un-irradiated control group gained an averageof 8.1% during the study, as compared with a mean loss of 0.8% in theplacebo group. In the groups receiving eritoran, a mean weight gain of4.0% was seen in the group receiving 1 mg/kg as compared with a net lossof 0.2% in the group receiving 10 mg/kg. The results of this analysisfor the three groups receiving radiation are shown in FIG. 12. Therewere no significant differences between these three groups (P=0.261).When compared against the un-irradiated controls, there were significantdifferences between the un-irradiated group and the radiated groupsreceiving placebo (P<0.001) and eritoran 10 mg/kg (P<0.001).

5.3 Tongue Histology

Each tongue was processed for routine hematoxylin and eosin histology.Because of several technical reasons, a total of 44 samples wereevaluated. Of these 44 samples, 6 were in the un-irradiated controlgroup, 12 were in the placebo group (6 each on days 6 and 10), 14 werein the eritoran 1 mg/kg treated group (7 each on days 6 and 10), and 12were in the eritoran 10 mg/kg treated group (7 on day 6, and 5 on day10).

The most common overall observation over the entire data set was normalor essentially normal. This was used in the description of 14 samples, 4of which were in the un-irradiated control group. Normal was also usedto describe 6 of the 14 samples in the 1 mg/mg eritoran treated group(2/7 samples from day 6 and 4/7 samples from day 10), and 3 of the 12samples in the eritoran 10 mg/kg treated group (1/7 samples from day 6and 2/5 samples from day 10). Only one of the 12 samples from theplacebo treated group was described as normal (a day 6 sample).Hyperkeratosis was also seen in 14 of the samples, none of which were inthe un-irradiated control group. Hyperkeratosis was most commonly seenin samples from the eritoran 10 mg/kg treated group, where it wasapplied to 7 of the 12 samples (3/7 at day 6 and 4/5 at day 10).Hyperkeratosis was seen in 5 of the 14 samples in the eritoran 1 mg/kgtreated group (1/7 at day 6 and 4/7 on day 10). Only 2 samples in theplacebo group were seen with hyperkeratosis, one at each time-point.Epithelial hyperplasia was seen in only 5 samples, however 4 of thesesamples were in the eritoran 10 mg/kg treatment group (2 at eachtime-point) and the fifth was in the placebo group (day 6). Theseobservations seem to indicate a substantial improvement in both eritorantreatment groups relative to the placebo controls, with the high dosetreatment group (10 mg/kg) showing a tendency to hyperplasia andhyperkeratosis.

Connective tissue damage or disruption was seen in a total of 11samples, 8 of which were in the placebo treated group (3/6 on day 6 and5/6 on day 10), 2 were in the eritoran 1 mg/kg treated group (both day6), 1 was in the eritoran 10 mg/kg treated group (day 6). Loss or breakin the epithelium was noted in 7 samples, and epithelial atrophy wasnoted in an additional 5 samples. Of these 12 samples with epithelialdamage, 5 were in the placebo treated group (2 on day 6 and 3 on day10), 5 were in the eritoran 1 mg/kg treated group (all on day 6), and 2were in the eritoran 10 mg/kg group (both on day 6). Increasedcellularity was seen in 10 samples, 2 in the placebo group (one each onday 6 and day 10), 5 in the eritoran 1 mg/kg treated group (one on day 6and 4 on day 10), and 3 in the eritoran 10 mg/kg treated group (one onday 6 and 2 on day 10). Two types of infiltrate were observed, roundcell or lymphocytic infiltrates were noted in 8 samples, were evenlydistributed among the groups and time-points, and were seen in one ofthe 6 un-irradiated controls. Mast cell infiltrates were observed in 9samples, 7 of which were in the placebo treated group (5 on day 6, and 2on day 10), and the other 2 samples were in the eritoran 10 mg/kgtreated group, day 6 time-point. The other observations regardingvasodilation and increased vascularity were evenly distributed or toorarely seen to show any meaningful differences between the treatmentgroups. These observations indicate that eritoran treatment results inimproved tongue histology, as shown by decreased radiation-inducedconnective tissue damage and mast cell infiltration.

5.3.1 Thickness of the Epithelial Surface on the Dorsal and VentralSurfaces of the Tongue

Each sample was evaluated for the minimum and maximum number ofepithelial cell layers on the dorsal and ventral surfaces of the tongue.From these numbers, mean minimum and maximum thickness was calculatedfor each treatment group at each time-point. For the dorsal surface ofthe tongue, the mean minimum number of cell layers in the un-irradiatedcontrols was 6 cell layers. In the placebo control treatment group, themean number of cell layers was 2.7 on day 6 and 2.0 on day 10. In theeritoran treated groups, the mean minimum number of cell layers was 2.8(day 6) and 3.8 (day 10) in the 1 mg/kg group and 3.8 (day 6) and 3.3(day 10) in the 10 mg/kg group. These data are shown in FIG. 13. Themean maximum number of epithelial cell layers on the dorsal surface inthe un-irradiated controls was 8. In the placebo control treatmentgroup, the mean number of cell layers was 4.8 on day 6 and 3.5 on day10. In the eritoran treated groups, the mean minimum number of celllayers was 5.3 (day 6) and 6.4 (day 10) in the 1 mg/kg group, and 6.2(day 6) and 6.1(day 10) in the 10 mg/kg group. These data are shown inFIG. 14. On the ventral surface, the mean minimum number of cell layersin the un-irradiated controls was 4 cell layers. In the placebo controltreatment group, the mean number of cell layers was 1.7 on day 6 and 0.9on day 10. In the eritoran treated groups, the mean minimum number ofcell layers was 2.0 (day 6) and 2.8 (day 10) in the 1 mg/kg group, and3.0 (day 6 and day 10) in the 10 mg/kg group. These data are shown inFIG. 15. The mean maximum number of epithelial cell layers on theventral surface in the un-irradiated controls was 6. In the placebocontrol treatment group, the mean number of cell layers was 4.2 on day 6and 2.4 on day 10. In the eritoran treated groups, the mean minimumnumber of cell layers was 3.7 (day 6) and 4.6 (day 10) in the 1 mg/kggroup and 5.8 (day 6) and 5.9 (day 10) in the 10 mg/kg group. These dataare shown in FIG. 16. These observations indicate that eritoran seems toprotect the epithelial cell layer, with the 10 mg/kg group showingslightly greater protection that the 1 mg/kg group, particularly on theventral surface.

6. Conclusions

1. Significant mortality was seen during this study, but this excessmortality was not associated with any one treatment group.

2. No statistically significant differences in weight gain were seenbetween the three irradiated treatment groups.

3. Both groups treated with eritoran showed improvement in tonguehistology relative to the placebo treated control group, as determinedby the number of samples described as normal, increases in epithelialhyperplasia and hyperkeratosis, and decreases in connective tissuedamage and mast cell infiltrates.

4. Although both groups treated with eritoran showed improvements intongue histology, there were distinct differences in the descriptivehistology between the 1 mg/kg and 10 mg/kg groups, although it is notclear which dose showed the greater improvement.

Example III 1. Introduction 1.1 Rationale

As discussed above, two strains of C3H mice (C3H/HeJ and C3H/HeOuJ)differ from one another by the presence or absence of the LPS receptorTLR4 (present in the C3H/HeOuJ strain), and the C3H/HeOuJ strain aresusceptible to oral mucositis induced by focal radiation to the snout,while the C3H/HeJ strain are relatively resistant to radiation inducedmucositis. Further as described above, evaluation of thepro-inflammatory cytokines in these animals shows that the induction ofthese cytokines via the LPS receptor (TLR4) in the C3H/HeOuJ mice mayplay a role in the development of oral mucositis. The experimentsdescribed in Example II demonstrated the efficacy of eritoran in a modelof oral mucositis. The study described below identifies optimal dosingschedules for eritoran.

1.2 Acute Snout Radiation Model

The acute mouse snout radiation model has been used to determine theradio-protective properties of experimental compounds. The course oforal mucositis in this model is well defined and results in peakmucositis 10-12 days following radiation. The acute model has littlesystemic toxicity, resulting in relatively few radiation induced animaldeaths. In this study, a dose of 30 Gy was used to induce oralmucositis.

2. Study Objective and Summary 2.1 Study Objective

The objective of this study was to examine the effect of scheduling oferitoran, administered intravenously, on the severity and duration oforal mucositis induced by radiation. Mucositis was induced using anacute radiation dose of 30 Gy directed to the mouse snout. At 10 daysafter radiation, groups of four mice from each treatment group weresacrificed. At the time of sacrifice, the tongues were removed and fixedin formalin for subsequent histological analysis. At the time ofsacrifice, blood was taken from each animal and serum was prepared forsubsequent cytokine analysis. These samples were used for themeasurement of serum Tumor Necrosis Factor (TNF-α), Interleukin-6(IL-6), and Serum Amyloid A (SAA) levels.

2.2 Study Summary

Sixty (60) C3H/HeOuJ mice were obtained from Jackson Laboratories. Theseanimals were shipped with jugular cannulae already implanted. Theanimals were randomly divided into 6 groups of 10 animals per group asdescribed in Table 4.

3. Study Design

Sixty (60) male C3H/HeOuJ mice aged 6-7 weeks and weighing approximately22 g were used. There were five (5) treatment groups of ten (10) animalseach, and a control group of ten (10) animals, which received noradiation. Beginning on day 0, 2 hours or less before radiation, animalsin groups 1-6 were dosed with either placebo or eritoran 10 mg/kg asdetailed in Table 4. Dosing continued twice daily from the day ofradiation (day 0) until day 9. Animals in groups 1 and 2 receivedplacebo throughout the dosing period. Animals in group 3 receivederitoran at 10 mg/kg for the entire dosing period. Animals in group 4received eritoran at 10 mg/kg twice daily from day 0 until day 3, andthen placebo twice daily until the end of the dosing period. Animals ingroup 5 received placebo twice daily from day 0 until day 2, theneritoran 10 mg/kg twice daily from day 3 until day 6, and then placebotwice daily until the end of the dosing period. Animals in group 6received placebo twice daily from day 0 until day 5, then eritoran 10mg/kg twice daily until the end of the dosing period. All drug andplacebo administration was via intravenous via jugular cannula.

TABLE 4 Allocation by experimental group Number of Dose Group AnimalsTreatment Eritoran Placebo Volume 1 10 Male No Radiation Days 0-9 0.1 mLPlacebo 2 10 Male Placebo Days 0-9 0.1 mL 3 10 Male eritoran days 0-9Days 0-9 0.1 mL 10 mg/kg bid 4 10 Male eritoran days 0-3 Days 0-3 Days4-9 0.1 mL 10 mg/kg bid 5 10 Male eritoran days 3-6 Days 3-6 Days 0-2 &0.1 mL 10 mg/kg bid Days 7-9 6 10 Male eritoran days 6-9 Days 6-9 Days0-5 0.1 mL 10 mg/kg bidEvery day for the period of the study (day 0 to day 10), each animal wasweighed to an accuracy of 0.1 g. At 10 days after radiation, all animalswere sacrificed and the tongues taken for histological analysis. Bloodwas taken at the time of sacrifice and serum was stored at −80° C.

4. Material and Methods 4.1 Animals

C3H/HeOuJ mice (Jackson Laboratories), aged 5 to 6 weeks with bodyweights of 23.2 g, were used. Animals had jugular cannulas installed byJackson Laboratories prior to shipment, and were individually numberedusing an ear punch and individually housed. Animals were acclimatizedprior to study commencement. During this period of at least 2 days, theanimals were observed daily in order to reject animals that presented inpoor condition.

4.2 Housing.

The study was performed in animal rooms as described above in section4.2 of Example I.

4.3 Diet

Animals were fed with Labdiet® 5061 sterile irradiated rodent chow andwater was provided ad libitum.

4.4 Animal Randomization and Allocations

Mice were randomly and prospectively divided into three (3) treatmentgroups prior to irradiation. Each animal was identified by an ear punchcorresponding to an individual number. A cage card was used to identifyeach cage or label marked with the study number, treatment group number,and animal numbers.

4.5 Radiation

Machine calibration was checked within two weeks of the onset of thisstudy. A single dose of radiation (30 Gy/dose) was administered to allanimals in groups 1 and 2 on day 0. Radiation was generated with a 160kilovolt potential (15-ma) source at a focal distance of 50 cm, hardenedwith a 0.35 mm Cu filtration system. Irradiation was done at a rate of121.5 cGy/minute. Animals were anesthetized prior to radiation, andplaced under lead shielding such that only the snout is exposed

4.6 Tissue Collection and Analysis 4.6.1 Histology

Histological samples were fixed in 10% formaldehyde in saline andprocessed for paraffin histology using standard techniques. Slides werestained with hematoxylin and eosin (H&E).

4.6.2 Cytokine ELISA

Enzyme linked immunosorbent assays (ELISAs) were performed for cytokinesTNF-α and IL-6 using kits purchased from R and D systems. Determinationof serum amyloid A was performed using an ELISA kit from BiosourceInternational. These kits were used in accordance with themanufacturer's instructions. All determinations were made in duplicateon serum samples stored at −80° C. Samples were run in duplicate in allthree assays, and if insufficient serum had been collected to run IL-6,SAA, and TNF-α assays, samples were diluted 1:4. All assays wereperformed using 50 μL of sample per well.

4.7 Assessment of Results

Statistical differences between treatment groups were determined usingOne Way ANOVA. Body weights are evaluated for differences between thetreatment groups.

5. Results and Discussion 5.1 Survival

A total of 108 cannulated animals were used in this study. Due to thelimited availability of the C3H/HeOuJ mice, these animals were processedin 3 groups over a period of 6 weeks. 57 of these mice survived untilday 10. Of the 51 mice that did not survive until day 10, 21 died orwere euthanized on day 0, 11 due to anesthesia and radiation relatedissues, and 10 due to problems with the cannula (died after initialinjection due to presumed clot, cannula not patent, or cannula pulledout). Of the remaining 30 animals that died or were enthanized duringthe study, 2 died on day 1, 5 on day 2, 4 on day 3, 7 on day 4, 3 eachon days 5 and 6, 1 each on days 7 and 8, and 2 each on days 9 and 10.The distribution of deaths by group was relatively equal. Nine (9)deaths were observed in each of the un-irradiated control group and thevehicle control group. Seven (7) deaths were observed in each of thegroups treated with eritoran 10 mg/kg from days 0-10 or days 0-3. Ninedeaths were observed in the group treated with eritoran 10 mg/kg, fromday 3 until day 6, and 10 deaths were observed in the group treated witheritoran 10 mg/kg, from day 6 until day 9.

5.2 Weights (FIGS. 17, 18, 19, and 20)

The mean percentage weight gain for each group for each day of the studyis shown in FIG. 17. The un-irradiated control group gained and averageof 3.2% during the study, as compared with a mean loss of 12.1% in theplacebo group. In the groups receiving eritoran at 10 mg/kg, a meanweight loss of 7.8% was seen in the group treated on days 0-10 ascompared with a net loss of 2.2% in the group treated on days 0 to 3, anet loss of 7.3% in the group treated on days 3 to 6, and a net loss of8.9% for the group treated on days 6 to 9. To determine whether thedifferences observed in weight change were significant, a One-Way ANOVAon the mean Area Under the Curve (AUC) data was performed. The resultsof this analysis are shown in FIG. 18. Three groups receiving radiationwere significantly different from the un-irradiated controls, theplacebo group (P<0.001), the group treated with eritoran from day 0until day 10 (P=0.014), and the group treated with eritoran from day 6to day 9 (P=0.025). The groups treated with eritoran from day 0 untilday 3 or from day 3 to day 6 were not significantly different than theun-irradiated controls. However, the group treated with eritoran fromday 0 until day 3 had significantly less weight loss than the placebocontrols (P=0.030). The weight data was reanalyzed with the data fromall animals dying during the study removed. The results of this analysisare shown in FIGS. 19 and 20. There was little change in the results ofthe One-Way ANOVA analysis, except that the group treated with eritoranfrom day 0 until day 9 was not significantly different from theun-irradiated controls in this analysis.

5.3 Tongue Histology

Each tongue was processed for routine hematoxylin and eosin histologyand slides were reviewed in a blinded manner. A total of 57 samples wereevaluated, and of these, 9 were in the un-irradiated control group, 9were in the placebo group, 11 were in the group treated with eritoran at10 mg/kg from day 0 to day 9, 11 were in the group treated with eritoranat 10 mg/kg from day 0 to day 3, 9 were in the group treated witheritoran at 10 mg/kg from day 3 to day 6, and 9 were in the grouptreated with eritoran at 10 mg/kg from day 6 to day 9. Three sectionsfrom each sample were evaluated for the following parameters: epitheialscore, connective tissue score, inflammation score, mitoses per 10 highpower fields (hpf), percent ulceration, number of inflammatory cells per10 hpf (percent neutrophils, lymphocytes, and monocytes/macrophage), thenumber of small, medium, and large blood vessels per 10 hpf, and thenumber of mast cells per 10 hpf.

5.3.1 Epithelial Score

Epithelial histology was scored on a 4 point 0-3 scale as outlined insection 4.7.1. These scores are shown in FIG. 21. The un-irradiatedanimals all had scores of 0. The placebo control group had a mean scoreof 1.1, as did the group treated with eritoran at 10 mg/kg from day 0 today 9. The group treated with eritoran at 10 mg/kg from day 0 to day 3had a mean score of 0.45. The group treated with eritoran at 10 mg/kgfrom day 3 to day 6 had a mean score of 0.89. The group treated witheritoran at 10 mg/kg from day 6 to day 9 had a mean score of 0.75.

5.3.2 Connective Tissue Score

Connective histology was scored on a 4 point 0-3 scale as outlined insection 4.7.1. These scores are shown in FIG. 22. The un-irradiatedanimals all had scores of 0. The placebo control group had a mean scoreof 0.4, the group treated with eritoran at 10 mg/kg from day 0 to day 9had a mean score of 0.6. The group treated with eritoran at 10 mg/kgfrom day 0 to day 3 had a mean score of 0.4. The group treated witheritoran at 10 mg/kg from day 3 to day 6 had a mean score of 0.6. Thegroup treated with eritoran at 10 mg/kg from day 6 to day 9 had a meanscore of 0.8.

5.3.3 Inflammation Score

Inflammation was scored on a 4 point 0-3 scale as outlined in section4.7.1. These scores are shown in FIG. 23. The un-irradiated animals allhad scores of 0. The placebo control group had a mean score of 0.4, thegroup treated with eritoran at 10 mg/kg from day 0 to day 9 had a meanscore of 0.5. The group treated with eritoran at 10 mg/kg from day 0 today 3 had a mean score of 0.4. The group treated with eritoran at 10mg/kg from day 3 to day 6 had a mean score of 0.6. The group treatedwith eritoran at 10 mg/kg from day 6 to day 9 had a mean score of 0.8.

5.3.4 Number of Mitoses

The number of mitoses was counted in 10 high power fields (hpf). Thesedata are shown in FIG. 24. The un-irradiated animals had an average of1.2 mitoses per 10 hpf. The placebo control group had an average of 3.9mitoses per 10 hpf. The group treated with eritoran at 10 mg/kg from day0 to day 9 had an average of 2.3 mitoses per 10 hpf. The group treatedwith eritoran at 10 mg/kg from day 0 to day 3 had an average of 1.5mitoses per 10 hpf. The group treated with eritoran at 10 mg/kg from day3 to day 6 had an average of 1.6 mitoses per 10 hpf. The group treatedwith eritoran at 10 mg/kg from day 6 to day 9 had an average of 1.5mitoses per 10 hpf.

5.3.5 Percent Ulceration

The percentage ulceration was estimated for each sample. These data areshown in FIG. 25. The un-irradiated animals had no ulceration. Theplacebo control group had mean ulceration of 13.3%. The group treatedwith eritoran at 10 mg/kg from day 0 to day 9 had mean ulceration of13.2%. The group treated with eritoran at 10 mg/kg from day 0 to day 3had mean ulceration of 2.7%. The group treated with eritoran at 10 mg/kgfrom day 3 to day 6 had mean ulceration of 16.7%. The group treated witheritoran at 10 mg/kg from day 6 to day 9 had mean ulceration of 10.0%.

5.3.6 Inflammatory Cell Infiltrates

The inflammatory cell infiltrate present in each sample was enumeratedby counting the total number of inflammatory cells per 10 hpf, andevaluated for cell type by estimating the percentage of cells within theinfiltrate that were neutrophils, lymphocytes, or monocytes/macrophage.The numbers of inflammatory cell data are shown in FIG. 26, the percentneutrophils in FIG. 27, the percent lymphocytes in FIG. 28, and thepercent monocytes/macrophage in FIG. 29. The un-irradiated animals hadan average of 9.3 cells per 10 hpf, with an average composition of 98.9%lymphocytes and 1.1% monocytes/macrophage, with no neutrophils seen. Theplacebo control group had an average of 44.9 cells per 10 hpf, with anaverage composition of 10.6% neutrophils, 86.7% lymphocytes, and 2.8%monocytes/macrophage. The group treated with eritoran at 10 mg/kg fromday 0 to day 9 had an average of 43.6 cells per 10 hpf, with an averagecomposition of 13.6% neutrophils, 82.7% lymphocytes, and 4.5%monocytes/macrophage. The group treated with eritoran at 10 mg/kg fromday 0 to day 3 had an average of 33.3 cells per 10 hpf, with an averagecomposition of 6.4% neutrophils, 93.2% lymphocytes, and 0.5%monocytes/macrophage. The group treated with eritoran at 10 mg/kg fromday 3 to day 6 had an average of 31.5 cells per 10 hpf, with an averagecomposition of 7.2% neutrophils, 91.1% lymphocytes, and 1.1%monocytes/macrophage. The group treated with eritoran at 10 mg/kg fromday 6 to day 9 had an average of 52.1 cells per 10 hpf, with an averagecomposition of 8% neutrophils, 91.0% lymphocytes, and 1.0%monocytes/macrophage.

5.3.7 Blood Vessels

The number of blood vessels present in each sample was quantified bycounting the total number of blood vessels in 10 hpf, and evaluated forvessel size by counting the number of small, medium, and large vesselsin this sample. These data are shown in FIGS. 30-32. The un-irradiatedanimals had an average of 5.6 blood vessels per 10 hpf, with an averagecomposition of 63.3% small, 20.7% medium, and 16.0% large vessels seen.The placebo control group had an average of 8.8 blood vessels per 10hpf, with an average composition of 63.9% small, 22.3% medium, and 13.9%large vessels. The group treated with eritoran at 10 mg/kg from day 0 today 9 had an average of 9.4 blood vessels per 10 hpf, with an averagecomposition of 74.6% small, 16.1% medium, and 9.3% large vessels seen.The group treated with eritoran at 10 mg/kg from day 0 to day 3 had anaverage of 8.2 blood vessels per 10 hpf, with an average composition of72.5% small, 14.9% medium, and 12.6% large vessels. The group treatedwith eritoran at 10 mg/kg from day 3 to day 6 had an average of 7.0blood vessels per 10 hpf, with an average composition of 67.9% small,20.5% medium, and 11.6% large vessels. The group treated with eritoranat 10 mg/kg from day 6 to day 9 had an average of 7.5 blood vessels per10 hpf, with an average composition of 72.1% small, 17.3% medium, and10.6% large vessels.

5.3.8. Mast Cells

The number of mast cells present in each sample was determined bycounting the number cells per 10 hpf. These data are shown in FIG. 33.The un-irradiated animals had 23.7 mast cells per 10 hpf. The placebocontrol group had 26 mast cells per 10 hpf. The group treated witheritoran at 10 mg/kg from day 0 to day 9 had 18.4 mast cells per 10 hpf.The group treated with eritoran at 10 mg/kg from day 0 to day 3 had 24.4mast cells per 10 hpf. The group treated with eritoran at 10 mg/kg fromday 3 to day 6 had 24.2 mast cells per 10 hpf. The group treated witheritoran at 10 mg/kg from day 6 to day 9 had 24.5 mast cells per 10 hpf.

5.4 Serum Cytokine Levels

Serum levels of TNF-α, IL-6, and SAA were measured using commerciallyavailable ELISA kits.

5.4.1 Serum TNF-α Levels

The un-irradiated animals had serum TNF-α levels of 43.0 pg/mL. Theplacebo control group had mean serum TNF-α levels of 63.8 pg/mL. Thegroup treated with eritoran at 10 mg/kg from day 0 to day 9 had meanserum TNF-α levels of 62.0 pg/mL. The group treated with eritoran at 10mg/kg from day 0 to day 3 had mean serum TNF-α levels of 20.3 pg/mL. Thegroup treated with eritoran at 10 mg/kg from day 3 to day 6 had meanserum TNF-α levels of 40.2 pg/mL. The group treated with eritoran at 10mg/kg from day 6 to day 9 had mean serum TNF-α levels of 119.1 pg/mL.These data are shown in FIG. 34.

5.4.2 Serum IL-6 Levels

The un-irradiated animals had serum IL-6 levels of 48.7 pg/mL. Theplacebo control group had mean serum IL-6 levels of 154.2 pg/mL. Thegroup treated with eritoran at 10 mg/kg from day 0 to day 9 had meanserum IL-6 levels of 85.6 pg/mL. The group treated with eritoran at 10mg/kg from day 0 to day 3 had mean serum IL-6 levels of 19.7 pg/mL. Thegroup treated with eritoran at 10 mg/kg from day 3 to day 6 had meanserum IL-6 levels of 50.4 pg/mL. The group treated with eritoran at 10mg/kg from day 6 to day 9 had mean serum IL-6 levels of 119.3 pg/mL.These data are shown in FIG. 35.

5.4.3 Serum SAA Levels

The un-irradiated animals had serum SAA levels of 597 μg/mL. The placebocontrol group had mean serum SAA levels of 427 μg/mL. The group treatedwith eritoran at 10 mg/kg from day 0 to day 9 had mean serum SAA levelsof 344 μg/mL. The group treated with eritoran at 10 mg/kg from day 0 today 3 had mean serum SAA levels of 279 μg/mL. The group treated witheritoran at 10 mg/kg from day 3 to day 6 had mean serum SAA levels of475 μg/mL. The group treated with eritoran at 10 mg/kg from day 6 to day9 had mean serum SAA levels of 652 μg/mL. These data are shown in FIG.36.

6. Conclusions

1. There was no evidence of toxicity with eritoran in the mortality orweight loss data from this study. As with previous studies, mortalitywas high, but evenly distributed across groups.

2. Mice treated with eritoran on days 0-3 showed a significantimprovement in weight loss relative to the placebo treated controlgroup.

3. The levels of oral mucositis observed in the placebo treated controlmice were lower than anticipated, and made it difficult to assess theimpact of eritoran on the levels of oral mucositis seen.

4. Possibly due to the relatively low levels of mucositis seen in theplacebo control group, little effect was seen in the group treated witheritoran at 10 mg/kg from day 0 to day 9, in contrast to previousobservations of efficacy with this treatment protocol.

5. Among the groups receiving radiation, the group treated with eritoranon days 0-3 had the lowest epithelial score, connective tissue score,and percent ulceration, indicating that it had suffered less damage thanother groups. This group also had the lowest inflammation score and wassecond lowest in the number of inflammatory cells and mitoses.

6. Among the groups receiving radiation, the group treated with eritoranon days 0-3 had the lowest serum levels of TNF-α, IL-6, and SAA, showingthe efficacy of this regimen in reducing inflammatory responses.

1. A method of reducing the severity of mucositis in a patient, themethod comprising the step of administering to the patient a compositioncomprising a compound that blocks activation of toll-like receptor
 4. 2.The method of claim 1, wherein the compound is a lipid A analog.
 3. Themethod of claim 2, wherein the lipid A analog is within the formula:

where R¹ is selected from the group consisting of:

where each J, K, and Q, independently, is straight or branched C1 to C15alkyl; L is O, NH, or CH₂; M is O or NH; and G is NH, O, S, SO, or SO₂;R² is straight or branched C5 to C15 alkyl; R³ is selected from thegroup consisting of straight or branched C5 to C18 alkyl,

where E is NH, O, S, SO, or SO₂; each A, B, and D, independently, isstraight or branched C1 to C15 alkyl; R⁴ is selected from the groupconsisting of straight or branched C4 to C20 alkyl, and

where each U and V, independently, is straight or branched C2 to C15alkyl and W is hydrogen or straight or branched C1 to C5 alkyl; R_(A) isR⁵ or R⁵—O—CH₂—, R⁵ being selected from the group consisting ofhydrogen, J′, -J′-OH, -J′-O—K′, -J′-O—K′—OH, and -J′-O—PO(OH)₂, whereeach J′ and K′, independently, is straight or branched C1 to C5 alkyl;R⁶ is selected from the group consisting of hydroxy, halogen, C1 to C5alkoxy and C1 to C5 acyloxy; A¹ and A², independently, are selected fromthe group consisting of

where Z is straight or branched C1 to C10 alkyl; or a pharmaceuticallyacceptable salt or phosphate ester thereof.
 4. The method of claim 3,wherein the lipid A analog is of the structure:

or a pharmaceutically acceptable salt or phosphate ester thereof.
 5. Themethod of claim 4, wherein the lipid A analog is of the structure:

or a pharmaceutically acceptable salt or phosphate ester thereof.
 6. Themethod of claim 1, wherein the mucositis is oral mucositis.
 7. Themethod of claim 1, wherein the mucositis is of the gastrointestinaltract.
 8. The method of claim 1, wherein the patient has mucositis. 9.The method of claim 1, wherein the patient does not have, but is at riskof developing, mucositis.
 10. The method of claim 9, wherein developmentof mucositis is inhibited in the patient by administration of thecomposition.
 11. The method of claim 10, wherein development ofmucositis is prevented in the patient by administration of thecomposition.
 12. The method of claim 1, wherein the patient is a cancerpatient.
 13. The method of claim 1, wherein the patient has recentlybeen, will shortly be, or is currently subject to treatment with head orneck irradiation, or stem cell or bone marrow transplantation.
 14. Themethod of claim 1, wherein said administration step occurs prior to,concurrently with, or after a treatment that places the patient at riskof developing mucositis, or a combination thereof.
 15. The method ofclaim 14, wherein said administration step occurs prior to a treatmentthat places the patient at risk of developing mucositis.
 16. The methodof claim 14, wherein said administration step occurs concurrently with atreatment that places the patient at risk of developing mucositis. 17.The method of claim 14, wherein said administration step occurs aftertreatment that places the patient at risk of developing mucositis. 18.The method of claim 14, wherein said administration step occursconcurrently with a treatment that places the patient at risk ofdeveloping mucositis, further comprising a step of administering thecomposition at least once during days 0-3 after the treatment thatplaces the patient at risk of developing mucositis.
 19. The method ofclaim 14, wherein the treatment that places the patient at risk ofdeveloping mucositis comprises radiation therapy.
 20. The method ofclaim 14, wherein the treatment that places the patient at risk ofdeveloping mucositis comprises chemotherapy.
 21. The method of claim 1,wherein the composition is administered to the patient topically. 22.The method of claim 1, wherein the composition is administered to thepatient by intravenous infusion.
 23. The method of claim 1, furthercomprising the step of administering antimicrobial therapy to thepatient.
 24. The method of claim 23, wherein the antimicrobial therapyis antibiotic therapy.